1. Field of the Invention
This invention relates to an immunoassay of an analyte and materials used therein, and more particularly relates to a method and materials for immunoassay which does not require separation of bound and free fractions.
2. Description of the Prior Art
A variety of assay systems which are both rapid and sensitive has been developed to determine the concentration of a substance in a fluid. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ the antigen in labeled form, the labeled antigen often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous or homogeneous. Heterogeneous assays require a separation of bound tracer from free (unbound) tracer. Homogeneous assays do not require a separation step and thereby provide significant advantage in speed, convenience and ease of automation over heterogeneous assays.
Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. However, all RIA procedures require a separation step, since the parameter measured (nuclear decay) cannot be controlled by changing assay conditions or components. In addition, isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must be followed.
Enzymes have also been used as labels in immunoassay. Enzymeimmunoassay (EIA) may be homogeneous and does not require precautions against radioactivity. Conjugation of an enzyme with a protein is usually straightforward, and the resulting protein-enzyme conjugate is generally stable. However, EIA depends on the reaction of the enzyme conjugate with a substrate to produce a color which is measured, and thus requires the additional step of providing an enzyme substrate. In addition, sufficient time must be allowed for color development and an expensive spectrophotometer for measuring color change must be provided.
Some of the above disadvantages associated with RIA or EIA have been overcome by use of fluorochromes as labels in immunoassay. Fluoroimmunoassay (FIA) provides direct detection of the label and is readily adaptable to homogeneous assay procedures. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA or EIA, largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium.
The development of time-resolved fluoroimmunoassay (TR-FIA) has contributed to overcoming these problems, however, FIA methods in general require complex instrumentation to provide the incident radiation and separate it from the fluorescence emission to be measured.
Other immunoassay procedures have been developed in which the label is chemiluminescent. In chemiluminescence immunoassay (CIA), light is produced by a chemical reaction and external incident radiation is not required. U.S. Pat. No. 4,104,029 to Maier discloses CIA in which the chemiluminescence, preferably from luminol, is measured directly. U.S. Pat. No. 4,375,972 to Forgione, et al. discloses catalysis of luminol chemiluminescence by a metallo-porphyrin. In U.S. Pat. No. 4,238,195 to Boguslaski et al. the chemiluminescence is absorbed by a fluorochrome present in the assay medium and is emitted and measured as fluorescence emission. U.S. Pat, No. 4,372,745 to Mandle et al. discloses encapsulation of a fluorochrome, conjugation of the encapsulated fluorochrome to a component of an immunological reaction, and disruption of the capsule to free the fluorochrome prior to activation of the chemiluminescent reaction.
CIA and EIA are combined in U.S. Pat. No. 4,302,534 to Halmann et al. An enzyme conjugated to a component of an immunological reaction reacts with a substrate to produce light which is measured. In U.S. Pat. No. 4,492,751 to Boguslaski et al., the enzyme substrate rather than the enzyme is conjugated to the immunological component.
In U.S. Pat. No. 4,220,450 to Maggio, a chemiluminescent agent conjugated to a ligand and a quenching agent conjugated to an antiligand are brought into sufficiently close proximity by immunological binding that emission from the bound chemiluminescent agent is energy-transferred to the quenching agent and thereby modulated in a detectable way, as, for example, by signal reduction resulting from radiationless decay, or emission at a different wavelength.
Although the above methods have improved immunoassay, there is still a need for a procedure affording high sensitivity which can be carried out rapidly without requiring a separation of bound and free fractions.